As the amount of HCV in the body is limited and an in vitro proliferation system of HCV has not been established, an anti-serum for HCV by using native virus particles or purified virus proteins has not been obtained. The individuals produce different antibodies to the antigen in their serum and the epitopes of the antibodies are different individually. Furthermore, as in human serum, antibodies against substances other than HCV may be contained. We must judge the result of HCV antibody testing considering sufficiently the cross reactivity etc.
As antibodies may have not been produced immediately after HCV infection, the antibody to HCV proteins cannot be detected by antibody testing during the period called “the window period” before the emergence of antibodies in the blood. For the treatment of patients with hepatitis C, various interferons and ribavirin may be effective, but the antibody test alone is not sufficient for the selection of therapeutic regimens and for monitoring disease conditions of patients. Under these circumstances, attention has been focused on methods of detecting HCV antigens and genes including definitive diagnosis.
As methods of detecting the HCV gene, the nucleic acid amplification technique (NAT) and the DNA probe method are known and are widely used in clinical settings. Though the NAT technique is highly sensitive, it also has several difficult problems. For example, in order to carry out a polymerase chain reaction (PCR) method, reverse transcription into DNA is required because HCV is a RNA virus, and a loss may easily occur during transcription from RNA to DNA; contamination may easily occur; a large quantity of specimens cannot be processed at one time since special equipment for amplification etc. is required and the procedure is complicated; and the cost for testing is expensive, and the like. In other NAT's as well, special equipment for amplification etc. is required and the cost for testing is enormous. The DNA probe method is not sensitive enough and requires about 20 hours before results can be obtained (IGAKU TO YAKUGAKU (Japanese Journal of Medicine and Pharmacological Sciences) 31:961-970, 1994).
A major problem in methods for detecting the HCV gene is the poor stability of HCV RNA in test samples, and it has been pointed out that the determined values may decrease by the step of obtaining the serum, storage for medium to long periods, and freeze-thaw procedures. This reason, it is believed, is that any damage to the surface of HCV particles may trigger the ribonuclease (RNase) in the blood resulting in easy degradation of HCV RNA as well. Therefore, in order to perform the HCV RNA test routinely, we must take care the handling and storage.
As a method of detecting HCV, attention has been focused on the method of detecting the HCV antigen in addition to HCV RNA.
As methods of detecting the HCV antigen, as described in Japanese Unexamined Patent Publication (Kokai) No. 8-29427, attempts have been made to detect the core antigen in the serum using a monoclonal antibody that has a specificity for an epitope on the HCV core antigen. Though this method is inexpensive and offers results in a short period of time (about 3 hours) as compared to the PCR method, it has several serious problems in the practical use.
Thus, for the treatment of test samples (serum), it requires multi-stage processing such as the polyethylene glycol treatment (4° C. for 1 hour), centrifugation (15 minutes), supernatant removal, urea treatment, alkali treatment (30 minutes), and the addition of a neutralizing agent. As the procedure is complicated as described above, experience is required in order to obtain reproducible results and, besides, as treatment for two hours is required, a large quantity of samples cannot be processed at one time.
Because of the required steps of centrifugation, supernatant removal etc., it is very difficult to automate the procedure. In addition, it is 10 to 100-fold less sensitive than the PCR method, and, therefore, is low in clinical usefulness. According to Journal of Hepatology 23:42-45, 1995, the detection limit of this assay is in between 104-105 copies of HCV RNA/ml, and IGAKU TO YAKUGAKU (Japanese Journal of Medicine and Pharmacological Sciences) 6:1065-1070, 1996, describes that when the sera of 102 patients with chronic hepatitis C before treatment were measured, it only gave a positivity of 67% which is almost equal to the of the above-mentioned DNA probe method, in contrast to a 100% positivity by the competitive reverse transcription (CRT)-PCR method. Thus there was much room for improvement in terms of sensitivity.
The two methods for detecting the HCV antigen described in Japanese Patent No. 3176570 and Japanese Patent No. 3171827 do not require multi-stage processing, including centrifugation etc., which is a drawback of the above HCV antigen detection method in Japanese Unexamined Patent Publication (Kokai) No. 8-29427; are able to process HCV-containing specimens in only one or two steps for 30 minutes; and are able to detect HCV antigen at a high sensitivity. In addition, as the procedure has been simplified, it is possible to process a large quantity of samples at one time and reproducibility and accuracy are enhanced as well. Furthermore, this measurement method can give results at a lower cost and in a shorter period of time as compared to NAT.
However, it is necessary to develop a more clinically useful method which satisfies all aspects of high sensitivity, specificity, reproducibility, easy handling, a shorter processing time, and low cost.
Though the two methods of detecting the HCV antigen described in Japanese Patent No. 3176570 and Japanese Patent No. 3171827 require 30 minutes of sample processing time, a shorter processing time is further desired.
In particular, the point of care testing (POCT) is rapidly growing in the US where the streamlining of hospital management is in progress, and is expected to be introduced in Japan and other countries in the future. This is a method in which testing is performed at a site near the patient, the test result is immediately judged by the attending doctor, necessary treatment is administered immediately, and even the progress of treatment and monitoring of prognosis are made, and thus it is attracting attention as it could serve the purpose of greatly enhancing the quality of medical care. Compared to testing performed in the central clinical testing laboratory, POCT can reduce the cost related to sample transportation and testing equipment, and patients can receive testing and immediate treatment at one visit, thereby reducing the patient's burden. In order to respond to these needs in the future, sample processing time must be curtailed as much as possible.
Furthermore, as there are very few HCV particles and thus very little HCV antigen in the blood of patients with HCV infection, a more sensitive method of detection is desired.
Patent document 1: Japanese Unexamined Patent Publication (Kokai) No. 8-29427
Patent document 2: Japanese Patent No. 3176570
Patent document 3: Japanese Patent No. 3171827
Patent document 4: Japanese Unexamined Patent Publication (Kokai) No. 8-29427
Patent document 5: Japanese Patent No. 3176570
Non-patent document 1: IGAKU TO YAKUGAKU (Japanese Journal of Medicine and Pharmacological Sciences) 31:961-970, 1994
Non-patent document 2: Journal of Hepatology 23:42-45, 1995
Non-patent document 3: IGAKU TO YAKUGAKU (Japanese Journal of Medicine and Pharmacological Sciences) 6:1065-1070, 1996